Abstract: The human body is comprised of a large collection of diverse cell types, each providing a specialized and context-specific function. The establishment and maintenance of a cell's identity is largely driven by chromatin structure. Novel genomic tools, such as ATAC-seq, enable highly multiplexed readout of regulatory and functional elements in the genome; however, these approaches only infer the chromatin structure and regulation but do not capture how the structural architecture of the genome is related to regulation and function. Here, we propose to leverage the existing expertise between the Chen and Buenrostro labs by combining fluorescent in situ sequencing, super-resolution microscopy, and ATAC-seq to develop and validate in situ ATAC-seq and ATAC-ExSeq, novel technologies that directly sequence genomic regulatory elements within the context of the 3D architecture of the genome. Our approach will involve the development of molecular biology protocols in conjunction with complementary hardware and software platforms. We will utilize these technologies to characterize the structural organization of the epigenome as well as explore multi-modal in situ assays for transcription factor binding and RNA co-localization. These in situ technologies will represent a fundamentally new and enabling capability for studying chromatin architecture and single-cell epigenomics.